Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome

Background. From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings. The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions. Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas. © 2010 Moreira et al; licensee BioMed Central Ltd.

Solubilization of iron phosphate by free or immobilized spores and pellets of Aspergillus niger

The production of pellets by Aspergillus niger and the FePO 4 (FeP) solubilization through the use of free or immobilized spores and pellets were studied. The media Sabouraud, MS-FeP, MS-K 2HPO 4, Czapek and Malt were used for pellets yield. Enhanced growth and pellets production were found in the Sabouraud and MS-FeP than those produced by the other media. Pellets produced in Sabouraud were larger in size and formed well-defined spheres than those produced in MS-FeP. Pellets amounts varying from 2 to 8 mg mL, -1 were inoculated in the MS-FeP medium. The greatest quantity of solubilized FeP was found when 6 mg of pellets mL, -1 were used. While the free spores were the worst form used for FeP solubilization in culture medium, free pellets allowed for the production of the greatest quantities of soluble phosphate, even after repeated use of the pellets. In the soil, pellets solubilized similar quantities of FeP compared to the immobilized A. niger spores and can be used with advantage since they are easily produced. © 2010 Academic Journals Inc.

Bactérias e resposta inflamatória no útero de cadelas, após inseminação artificial

The present study aimed to obtain information about the uterine inflammatory response (number of polymorphonuclear neutrophilic granulocytes - PMNs) in bitches after artificial insemination (AI) and identify the uterine microflora present after the following treatments: insemination using semen with extender (n=6), insemination with fresh semen (n=6) and no inseminated (n=6). The percentage of PMNs on the endometrial surface and within histological sections was evaluated together with the presence of aerobic bacteria in the uterine lumen. For endometrial cytology, there was no significative difference on the number of inflammatory cells between bitches not inseminated (3.05 ± 1.74 PMNs) and those inseminated with fresh semen (3.55 ± 1.51 PMNs); There was a significative difference in both groups compared to the inseminated with semen plus extender (7.80 ± 1.67 PMNs) (p<0.05). Histology showed that there was no significative difference on the number of inflammatory cells between bitches not inseminated (87.72 ± 35.2 PMNs) and those inseminated with fresh semen (122.97 ± 43.31 PMNs); however, it was observed differences in both groups compared to those inseminated with semen plus extender (171.94 ± 42.74 PMNs) (p<0.05). Eight animals, randomly distributed in the groups, showed the presence of Staphylococcus sp and Proteus sp., in the microbiological exam. The extender for semen, with Tris, is a potent inducer of uterine inflammation, and positive uterine cultures may be obtained during estrus without inflammation or uterine infection.

Improving the affinity of tomato grafted on Solanum torvum using an intermediate rootstock

Most of the interspecific rootstocks (Lycopersicum esculentum × L. hirsutum) used in grafted Spanish tomato crops are resistant to Meloidogyne nematodes, but the 'Mi' resistance gene does not work well at high soil temperatures. Ralstonia solanacearum is a bacterial disease usual in tropical areas, but recently identified with low incidence in several European countries. This disease could be controlled by grafting tomato on Solanum torvum, which is also resistant to Meloidogyne. However, S. torvum and tomato have low grafting affinity, which could be improved using an intermediate rootstock. Some cultivars of eggplant have a relatively good affinity with tomato and complete affinity with S. torvum. In this study we compared two tomato cultivars (one resistant to Verticillium dalihae, Fusarium oxysporum v. lycopersici race 2 and Meloidogyne spp., and one non-resistant) grafted onto 'Beaufort' (Lycopersicum esculentum × L. hirsutum), 'Torvum Vigor' (Solanum torvum) and also with an intermediate grafting of eggplant ('Cristal') between tomato and S. torvum, with nongrafted plants as controls. This arrangement was carried out in two cropping cycles (winter-spring and summer-autumn). In both cycles, plants grafted onto S. torvum, both single or double grafted, yielded less than those grafted onto 'Beaufort' or nongrafted plants. In the spring cycle, no differences were found between single and double-grafted plants using S. torvum rootstocks, but in the autumn cycle double grafted plants had higher yields than the single grafted plants. The severity of nematode infections, in terms of reducing yields, and/or hypothetical infections of Ralstonia, will determine the utility of this technique in tomato production.

Contamination of cattle carcasses by Escherichia coli shiga like toxin with high antimicrobials resistence

During processing of cattle carcasses, contamination may occurs with the transfer of microbiota of animals feaces to carcasses. This contamination many times may be by Escherichia coli carriers of virulence factor as stx and eae genes being classified as Shiga like toxin. Shiga toxin-producing Escherichia coli (STEC) is recognized wordwide as human pathogen. A survey was performed to determine the sensibility profile to several antimicrobial drugs of STEC in carcasses obtained from an abattoir in Brazil between March 2008 and August at 2009. A total of 120 STEC were isolated. All isolates were confirmed as being E. coli by their biochemical analysis and submitted to polymerase chain reaction (PCR) for detection of stx, eae and ehly genes. No strains was isolated being carriers of ehly gene. The number of isolates carriers of eae gene were 48/120. The most frequent resistance was seen against cephalothin (84.0%), streptomycin (45.0%), nalidixic acid (42.0%) and tetracycline (20.0%). Multidrug resistance (MDR) to three or more antimicrobial agents was observed in 46 (38.3%) E. coli isolates. The findings of STEC and MRD show that cattle carcasses may be a reservoir of pathogenic bacterial for the consumer public. © 2011 Academic Journals.

Presence of a human diarrheagenic escherichia coli clone in captivity kept psittacidaes

Bacterial cultures of cloaca swabs from 86 captivity kept psittacidaes revealed 17 Escherichia coli bearing birds sharing strains which, on the basis of enterobacterial repetitive intergenic consensus (ERIC) PCR analysis, proved to be genetically similar. Further, triplex PCR specific for the genetic markers chuA, yjaA, and TSPE4.C2 was used to assign the strains to the E. coli reference collection (EcoR) B2 group. One strain of each, from the enteropathogenic (EPEC), enteroaggregative (EAEC) and Shiga toxin (STEC) E. coli pathovars were found among these isolates. © Marietto-Gonçalves et al.; Licensee Bentham Open.

Perfil Fermentativo e Valor Nutritivo de Silagem de Capim-Elefante Inoculada com Streptococcus Bovis

The experiment was developed to evaluate the effect of strains of Streptococcus bovis (HC5 and JB1) on pH, ammonia production and nutritional value in elephant-grass silages (Pennisetum purpureum Schum). The experimental design was entirely randomized, with three treatments: T1 - elephant- grass, T2 - grass-elephant inoculated with Streptococcus bovis HC5, T3 - elephant grass inoculated with Streptococcus bovis JB1, totaling five replicates per treatment. The smallest value of ammonia was observed in the silage treated with inoculante the base Streptococcus bovis JB1 (5.90% N-total). Inoculation increased the levels of lactic acid and decreased levels of acetic, propionic and butyric acids, and the silages inoculated with Streptococcus bovis JB1 and HC5 were those with the highest values of lactic acid. There were not statistical differences for the tenors of dry matter (MS) and crude protein (PB). For the values of the fibers, the grass-elephant silage inoculated with Streptococcus bovis JB1, it presented the smallest value of neutral detergent fiber (59.77 %) and Hemicellulose (26.71 %). The largest value of in vitro dry matter digestibility (DIVMS) was observed in the silage with Streptococcus bovis JB1, showing superiority of four percentile points (64.23 %) in relation to the treatment without bacterial inoculante (60.35 %). The use Streptococcus bovis JB1 was efficient in the process fermentativo besides promoting would get better in the quality of the fiber and DIVMS in the studied silage.

Presence and distribution of the endosymbiont Wolbachia among Solenopsis spp. (Hymenoptera: Formicidae) from Brazil and its evolutionary history

Wolbachia are intracellular bacteria that commonly infect arthropods. Its prevalence among ants of the genus Solenopsis is high. In the present study, the presence and distribution of these endosymbionts was examined among populations of Solenopsis spp. from Brazil. A phylogenetic analysis based on the wsp gene was conducted to infer the evolutionary history of Wolbachia infections within the populations surveyed. A high frequency of Wolbachia bacteria was observed among the genus Solenopsis, 51% of the colonies examined were infected. Incidence was higher in populations from southern Brazil. However, little genetic variability was found among different Wolbachia strains within supergroups A and B. Our findings also suggest that horizontal transmission events can occur through the social parasite S. daguerrei. © 2012 Elsevier Inc..

Atividade viral do vírus respiratório sincicial bovino (brsv) em bezerros leiteiros

The purpose of the present study was to evaluate the antimicrobial activity of propolis extracts diluted in different solvents against bacteria from Staphylococcus genus. The study was performed in the Immunology and Microbiology Laboratory from Universidade Federal do Vale do São Francisco. The propolis extracts were prepared using brown propolis diluted in different solvents such as chloroform, methanol, ethyl acetate and grain alcohol. In order to determine the antimicrobial potential of extracts, agar well diffusion method was used, with controls for each diluent. After that, Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) methods were used. All tests were performed in triplicate. In the agar well diffusion test, the measurements of the inhibition zone for propolis extract were as follows: grain alcohol and propolis (2.88mm), methanol and propolis (2.41mm), chloroform and propolis (2.40mm) and ethyl acetate and propolis (0.83mm). The MBC of propolis extracts in different solvents were 93.75 μg/mL for grain alcohol, 375 μg/mL for chloroform and methanol and 3,000 μg/ml for ethyl acetate. Statistically significant differences were achieved comparing the inhibition zones of propolis diluted in grain alcohol and ethyl acetate (2.88 and 0.83 mm, respectively). Considering the low cost of therapy and the activity of the propolis against caprine mastitis pathogens, other studies regarding in vivo activity and chemical characterization are necessary, in addition to evaluation of the toxicological aspects of propolis extracts.

Fermentation characteristics of several carbohydrate sources for dog diets using the in vitro gas production technique

Fermentable carbohydrates are an important part of the canine diet. They can improve gastrointestinal health by modifying gut microbial population and metabolic activity. The present study compared the fermentation characteristics and kinetic patterns of 10 carbohydrate sources using the in vitro gas production technique (IVGPT) with dog faecal inoculum. The substrates tested were: pure cellulose (PC), carboxymethylcellulose (CMC), sugar-cane fibre (SCF), beet pulp (BP), wheat bran (WB), fructooligosaccharides (FOS), inulin, yeast cell wall (YCW), ground psyllium seed (PS), pea hulls (PH). All substrates were incubated at 39°C under anaerobic conditions with faeces collected from dogs as microbial inoculum. Gas production of fermenting cultures was recorded and after 48 h, pH, shortchain fatty acids (SCFA) and organic matter disappearance (OMD) were determined. The results confirm high fermentation by dog faecal bacteria of FOS and inulin that produced high amounts of propionate and that underwent very rapid fermentation. Three substrates (SCF, CMC and PC) were not able to support bacterial growth, with low gas and SCFA production, and high BCFA formation. The PH and BP showed moderate OMD and SCFA production. Wheat bran B underwent rapid fermentation and generated a high proportion of butyrate. Psyllium seeds underwent slow fermentation with delayed gas production, supporting a high formation of SCFA, with an adequate amount of butyrate for bacterial growth while YCW, which showed a delayed fermentation, gave moderate SCFA production. The fermentation characteristics of PS and YCW suggest their potential use in promoting a more distal fermentation on intestinal tract. © Copyright S. Calabrò et al., 2013 Licensee PAGEPress, Italy.

Efeitos do extrato de parede de levedura em dieta seca sobre a Microbiologia, ácidos graxos de cadeia curta e redução do odor das fezes de gatos adultos

The aim of this study was to evaluate the effects of yeast cell wall extract (YCW) in dry diet on the fecal microbiota, concentration of short-chain fatty acids (SCFA) and on the odor reduction of cats feces. We used 20 animals of both sexes, randomly assigned to four treatments and five repetitions totaling 20 experimental units: 1) dry commercial diet (control); 2) control + 0.2%, 3) control + 0.4%, and 4) control + 0.6% of YCW in dry matter. Enterobacteriaceae and lactic acid bacteria, fecal concentration of acetic, propionic and butyric acids, ammonia nitrogen and sensory panel were performed. There were no significant differences (p> 0.05) for bacterial counts and the concentration of SCFA and ammonia, but in sensory panel a reduction in the odor of feces could be noted with the use of 0.2% of YCW. We concluded that the addition of up to 0.6% YCW had no effect on the microbiology and the concentration of fatty acids, but there is potential for its use as an additive because of the improvement in the odor of feces. However, further studies are needed to understand the mechanisms of action and the effects of prebiotics for domestic cats.

Keratinase production by new strain of Bacillus Amyloliquefaciens: Application of statistical experimental design for optimization of keratinase production

Feathers are rich in amino acids and can be employed as a dietary protein supplement for animal feed. Microbial degradation is an alternative technology for improving the nutritional value of feathers. Other potential applications of keratinase include use in the leather industry, detergents and medicine as well as the pharmaceutical for the treatment of acne, psoriasis and calluses. A new keratinolytic enzyme production bacterium was isolated from a poultry processing plant. To improve keratinase yield, statistically based experimental designs were applied to optimize three significant variables: temperature, substrate concentration (feathers) and agitation speed. Response surface methodology demonstrated an increase in keratinolytic activity at temperature, agitation speed and substrate concentration of 26.6°C, 150 rpm and 2%, respectively. Liquid chromatography revealed the release of amino acids in the Bacillus amyloliquefaciens culture broth, thereby demonstrating the potential of feather meal in the animal feed industry. © Global Science Publications.

Microbiological quality of organic and conventional vegetables sold in Brazil

While searching for healthier diets, people became more attentive to organic produce. Yet, organic foods may be more susceptible to microbiological contamination because of the use of organic fertilizers, a possible source of pathogenic bacteria. In this study, 130 samples of different organic and conventional vegetable varieties sold in Brazil were analyzed for mesophilic aerobic bacteria, yeasts and molds, total coliforms, Escherichia coli and Salmonella spp. Most of the mesophilic aerobic bacteria counts in organic and conventional vegetables ranged from 6 to 7 log10 CFU/g; most of the yeasts and molds counts ranged from 5 to 6 log10 CFU/g and most of the total coliforms counts ranged from 4 to 5 log10 CFU/g. E. coli was found in 41.5% of the organic and 40.0% of the conventional vegetables, and most samples had counts ranging from 1 to 2 log10 CFU/g. Salmonella spp. was not found in any sample. Comparative analyses of the microbial counts of organic and conventional vegetables showed that some organic varieties have greater counts. However, the global results show that this is not a trend. These results indicate the need of good farming practices, and proper sanitization before consumption, to ensure food quality and safety. © 2012 Elsevier Ltd.

Antibacterial activity of alkyl gallates against Xanthomonas citri subsp. citri

The plant-pathogenic bacterium Xanthomonas citri subsp. citri is the causal agent of Asiatic citrus canker, a seriousdisease that affects all the cultivars of citrus in subtropical citrus-producing areas worldwide. There is no curative treatment for citrus canker; thus, the eradication of infected plants constitutes the only effective control of the spread ofX. citri subsp. citri. Since the eradication program in the state of São Paulo, Brazil, is under threat, there is a clear risk of X. citri subsp. citri becoming endemic in the main orange-producing area in the world. Here we evaluated the potential use of alkyl gallates to prevent X. citri subsp. citri growth. These esters displayed a potent anti-X. citri subsp. citri activity similar to that of kanamycin (positive control), as evaluated by the resazurin microtiter assay (REMA). Thetreatment of X. citri subsp. citri cells with these compounds induced altered cell morphology, and investigations of the possible intracellular targets using X. citri subsp. citri strains labeled for the septum and centromere pointed to a commontarget involved in chromosome segregation and cell division. Finally, the artificial inoculation of citrus with X. citri subsp. citri cells pretreated with alkyl gallates showed that the bacterium loses the ability to colonize its host, which indicates the potential of these esters to protect citrus plants against X. citri subsp. citri infection. © 2013, American Society for Microbiology.

Rough virulent strain of Brucella ovis induces pro- and anti-inflammatory cytokines in reproductive tissues in experimentally infected rams

The ovine brucellosis caused by Brucella ovis has tropism for reproductive tissues but until now the mechanism of bacterial persistence is not understood. Cytokine expression profiles were studied for 8 months in rams after being experimentally infected with the rough virulent strain of B. ovis (R- B. ovis) to study the pathogenesis of B. ovis and immune mechanism possibly associated to bacteria tropism and persistence. The messenger RNA (mRNA) expression levels of interleukin-1α (IL-1α), IL-1β, IL-6, IL-10, IL-12, interferon-γ (INF-γ) and tumour necrosis factor-α (TNF-α) cytokines were quantified by real-time quantitative RT-PCR (qRT-PCR) in reproductive tissues (epididymus, testicles, ampolae, vesicular glands and bulbourethral glands), and non-reproductive (liver, spleen and kidneys) tissues at 30, 60, 120 and 240 days post infection (dpi). During the acute phase of infection at 30. dpi, the host immune response was most notable demonstrating an up-regulation of several cytokines in reproductive tissues, including the epididymus (IL-6, IL-1β and IL-1α), testicles (INF-γ and IL-12), bulbourethral glands (IL-6 and TNF-α) and ampolae (INF-γ, IL-10, IL-1β and IL-1α). During the development of infection, cytokine gene expression levels decreased, providing evidence of immunosuppression and evidence of immune evasion that favoured persistence of chronic R- B. ovis infection. During the chronic phase of R- B. ovis infection (120 and 240. dpi), cytokine production was down-regulated in the epididymus (IL-1β and IL-1α), testicles (INF-γ and IL-12), and ampolae (INF-γ, IL-10, IL-1β and IL-1α), with the exception of the bulbourethral glands (IL-6 and TNF-α) and epididymus (IL-6); in these tissues, R- B. ovis infection resulted in up-regulation of the pro-inflammatory cytokine IL-6. Herein, we report cytokine expression profiles in tissues of rams experimentally infected with the rough strain of B. ovis, which are associated with bacterial persistence and macrophage activation. © 2012 Elsevier B.V.